scholarly journals MUTATIONS IN radA ARE RESPONSIBLE FOR ACQUIRED RADIO-RESISTANCE IN AN Escherichia coli STRAIN

2021 ◽  
Author(s):  
David Alcántara Díaz ◽  
Jorge Humberto Serment Guerrero ◽  
Gerardo Aguirre Escalona ◽  
Jorge Tonatiuh Ayala Sumuano
Keyword(s):  
Escherichia Coli ◽  
Gamma Radiation ◽  
Uv Light ◽  
Point Mutations ◽  
Coli Strain ◽  
Normal Strain ◽  
Wild Type ◽  
Different Strains ◽  
Strain Ab1157 ◽  
Selection Of

When bacteria are exposed to chronic or cyclic irradiation with ultraviolet (UV) light, it is observed that their resistance to this agent is increased by the selection of advantageous mutations under those conditions. UV light produces different damages in DNA, the repair of which is necessary to maintain the integrity of the genome. However, some damages can lead to such mutations when they are not properly repaired. In an earlier work, five subcultures of a wild-type Escherichia coli strain (PQ30) were cyclically irradiated with UV and different strains resistant to UV light and gamma radiation were obtained. In a preliminary mapping, different genes involved in their resistance to radiation were identified. In one of these strains, designated as IN801, the radA gene, the product of which is involved in recombinational DNA repair, was identified. In this work, cells from another wild-type strain (AB1157) were transformed with a plasmid (pUC19) that carries the radA gene from either PQ30 or IN801, in order to establish whether the radio-resistant phenotype can be transferred to a normal strain. Only cells that received the IN801 radA gene showed increased resistance to UV and gamma radiation. Further radA sequencing showed that the gene of IN801 acquired two-point mutations that replace two amino acids in the RadA protein, which most likely changed its enzymatic activities. These results confirm that radA participates in the radiation resistance of IN801.

scholarly journals Altered Substrate Selection of the Melibiose Transporter (MelY) of Enterobacter cloacae Involving Point Mutations in Leu-88, Leu-91, and Ala-182 That Confer Enhanced Maltose Transport

2003 ◽  
Vol 185 (12) ◽  
pp. 3672-3677 ◽  
Author(s):  
Steven G. Shinnick ◽  
Stephanie A. Perez ◽  
Manuel F. Varela
Keyword(s):  
Escherichia Coli ◽  
Point Mutations ◽  
Enterobacter Cloacae ◽  
Substrate Selection ◽  
Coupled Transport ◽  
Wild Type ◽  
Transmembrane Segment ◽  
Maltose Transport ◽  
Site Classes ◽  
Selection Of

ABSTRACT We isolated mutants of Escherichia coli HS4006 containing the melibiose-H+ symporter (MelY) from Enterobacter cloacae that had enhanced fermentation on 1% maltose MacConkey plates. DNA sequencing revealed three site classes of mutations: L-88-P, L-91-P, and A-182-P. The mutants L-88-P and L-91-P had 3.6- and 5.1-fold greater maltose uptake than the wild type and enhanced apparent affinities for maltose. Energy-coupled transport was defective for melibiose accumulation, but detectable maltose accumulation for the mutants indicated that active transport is dependent upon the substrate transported through the carrier. We conclude that the residues Leu-88, Leu-91 (transmembrane segment 3 [TMS-3]), and Ala-182 (TMS-6) of MelY mediate sugar selection. These data represent the first MelY mutations that confer changes in sugar selection.

Sensitivity of normal and mutant strains of Escherichia coli to actinomycin-D

1972 ◽  
Vol 18 (6) ◽  
pp. 909-915 ◽  
Author(s):  
A. P. Singh ◽  
K.-J. Cheng ◽  
J. W. Costerton ◽  
E. S. Idziak ◽  
J. M. Ingram
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Wild Type Strain ◽  
Actinomycin D ◽  
Cytoplasmic Membrane ◽  
Cell Envelope ◽  
Coli Strain ◽  
Wild Type ◽  
Mutant Strains ◽  
In The Wild

The site of the cell barrier to actinomycin-D uptake was studied using a wild-type Escherichia coli strain P and its cell envelope-defective filamentous mutants, strains 6γ and 12γ, both of which 'leak' β-galactosidase and alkaline phosphatase into the medium during growth indicating both membrane and cell-wall defects. Actinomycin-D entered the cells of these two mutant strains as evidenced by the inhibition of both 14C-uracil incorporation and synthesis of the induced β-galactosidase system. Under similar conditions, no inhibition occurred in the wild-type strain and its sucrose-lysozyme prepared spheroplasts. Actinomycin-D did, however, inhibit the above-mentioned systems in the wild-type sucrose-lysozyme spheroplasts prepared in the presence of 2 mM EDTA. The experimental data indicate that although the cell wall may act as a primary barrier or sieve to actinomycin-D, the cytoplasmic membrane should be considered the final and determinative barrier to this antibiotic.

Homologous recombination involving a large heterology in Escherichia coli.

Genetics ◽  
1988 ◽  
Vol 119 (4) ◽  
pp. 759-769
Author(s):  
K Yamamoto ◽  
N Takahashi ◽  
H Yoshikura ◽  
I Kobayashi
Keyword(s):  
Escherichia Coli ◽  
Gene Conversion ◽  
Kanamycin Resistance ◽  
Coli Strain ◽  
The Other ◽  
Reca Gene ◽  
Inverted Repeats ◽  
Crossing Over ◽  
Wild Type ◽  
Parental Allele

Abstract Recombination between two different deletion alleles of a gene (neo) for neomycin and kanamycin resistance was studied in an Escherichia coli sbcA- recB-C- strain. The two homologous regions were in an inverted orientation on the same plasmid molecule. Kanamycin-resistant plasmids were selected and analyzed. The rate of recombination to form kanamycin-resistant plasmids was decreased by mutations in the recE, recF and recJ genes, but was not decreased by a mutation in the recA gene. It was found that these plasmids often possessed one wild-type kanamycin-resistant allele (neo+) while the other neo allele was still in its original (deletion) form. Among kanamycin-resistant plasmids with one wild-type and one parental allele it was often found that the region between the inverted repeats had been flipped (turned around) with respect to sites outside the inverted repeats. These results were interpreted as follows. Gene conversion, analogous to gene conversion in eukaryotic meiosis, is responsible for a unidirectional transfer of information from one neo deletion allele to the other. The flipping of the region between the inverted repeats is interpreted as analogous to the crossing over associated with gene conversion in eukaryotic meiosis. In contrast with a rec+ strain, these products cannot be explained by two rounds of reciprocal crossing over involving a dimeric form as an intermediate. In the accompanying paper we present evidence that gene conversion by double-strand gap repair takes place in the same E. coli strain.

Growth of Escherichia coli on glucosamine 6-phosphate: selection of a constitutive hexose phosphate transport system mutant

1978 ◽  
Vol 24 (3) ◽  
pp. 203-208
Author(s):  
George W. Dietz Jr.
Keyword(s):  
Escherichia Coli ◽  
Transport System ◽  
Phosphate Transport ◽  
Wild Type ◽  
Hexose Phosphate ◽  
Selection Of

Glucosamine 6-phosphate was found to be a substrate but not an inducer for the hexose phosphate transport system of Escherichia coli. Wild-type cells grow very poorly on glucosamine 6-phosphate. A mutant was selected that will grow rapidly on glucosamine 6-phosphate because it contains a constitutive hexose phosphate transport system.

scholarly journals REGULATION OF NEWLY EVOLVED ENZYMES. IV. DIRECTED EVOLUTION OF THE EBG REPRESSOR

Genetics ◽  
1978 ◽  
Vol 90 (4) ◽  
pp. 673-681
Author(s):  
Barry G Hall
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Enzyme Synthesis ◽  
Regulatory Function ◽  
Wild Type ◽  
Selection For ◽  
Selection Of

ABSTRACT In Escherichia coli, the wild-type repressor of ebg (evolved β-galactosidase) enzyme synthesis, specified by the ebgR  + gene, responds very weakly to lactulose (fructose-β-D-galactopyranoside). Selection for a functional repressor that responds strongly to lactulose as an inducer reveals the existence of ebgR+L mutants, which occur spontaneously at a frequency of about 2 x 10-10. ebgR+L mutants are pleiotropic in that they specify ebg repressor with a greatly increased response to lactulose, lactose, galactose-arabinoside and methyl-galactoside as inducers. Selection of ebgR+L mutants is discussed within the framework of directed evolution of a regulatory function.

scholarly journals Optimizations of expression and purification of recombinant HIV-1 CRF01_AE p24 protein in Escherichia coli for development of immunodiagnostic assay

2015 ◽  
Vol 24 (1) ◽  
pp. 14-8
Author(s):  
Ade P.R. Simaremare ◽  
Budiman Bela ◽  
Andi Yasmon ◽  
Fera Ibrahim
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
Coli Strain ◽  
Expression And Purification ◽  
Purification System ◽  
P24 Protein ◽  
Native Condition ◽  
Different Strains ◽  
Hiv 1 ◽  
Induction Temperature

Background: Conventional method for confirmation of HIV infection is Western blot. However, it has limitations because of contamination by human cellular antigen and genetic diversity among the HIV-1 subtypes that show indeterminate result and inaccuracy for the diagnosis of different strains. Most of Western blot developed are based on HIV-1 B subtype. In Indonesia HIV-1 CRF01_AE subtype is dominantly circulated. Therefore, we optimized the expression, purification of the recombinant HIV-1 CRF01_AE p24 protein for development of immunodiagnostic assay.Methods: Optimization of protein expression in Escherichia coli strain BL21CP was performed including induction time, isopropyl-1-thio-d-galactopyranoside (IPTG) and immidazole consentrations, and induction temperature. Purification of the recombinant p24 protein was used by using Ni-NTA (Qiagen) purification system in native condition. Results: Expression and purification of HIV-1 CRF01_AE p24 protein have been performed. Confirmation of the recombinant protein by Western blot showed the expression and purification of recombinant p24 protein has been optimized well and reactive with sera of patients with HIV-1 CRF01_AE subtype positive.Conclusion: The recombinant HIV-1 CRF01_AE p24 protein has been expressed and purified successfully, and it is potential to be used as antigen for immunodiagnostic assay.

scholarly journals Why Does Escherichia coli Grow More Slowly on Glucosamine than on N-Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

2005 ◽  
Vol 187 (9) ◽  
pp. 2974-2982 ◽  
Author(s):  
Laura I. Álvarez-Añorve ◽  
Mario L. Calcagno ◽  
Jacqueline Plumbridge
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Sugar Transport ◽  
Point Mutations ◽  
Sole Source ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Allosteric Activation

ABSTRACT Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

scholarly journals Inactivation of ibeA and ibeT Results in Decreased Expression of Type 1 Fimbriae in Extraintestinal Pathogenic Escherichia coli Strain BEN2908

2008 ◽  
Vol 76 (9) ◽  
pp. 4129-4136 ◽  
Author(s):  
Mélanie A. M. Cortes ◽  
Julien Gibon ◽  
Nathalie K. Chanteloup ◽  
Maryvonne Moulin-Schouleur ◽  
Philippe Gilot ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Wild Type Strain ◽  
Coli Strain ◽  
Wild Type ◽  
Adhesive Properties ◽  
Type 1 Fimbriae ◽  
Pathogenic Escherichia Coli ◽  
Genes Encoding

ABSTRACT IbeA in extraintestinal pathogenic Escherichia coli (ExPEC) strains was previously described for its role in invasion. Here we investigated the role of IbeA and IbeT, encoded by a gene located downstream of ibeA, in the adhesion of the avian ExPEC strain BEN2908 to human brain microvascular endothelial cells (HBMEC). The ΔibeA mutant was less adhesive to HBMEC than the wild-type strain BEN2908 was. Because strain BEN2908 also expresses type 1 fimbriae, we measured the adhesion specifically due to IbeA by comparing the adhesive properties of a Δfim derivative of strain BEN2908 to those of a double Δfim ΔibeA mutant. No differences were observed, indicating that the reduction of adhesion in BEN2908 ΔibeA could be due to a decrease in type 1 fimbria expression. We indeed showed that the decreased adhesion of BEN2908 ΔibeA was correlated with a decrease in type 1 fimbria expression. Accordingly, more bacteria had a fim promoter orientated in the off position in a culture of BEN2908 ΔibeA than in a culture of BEN2908. Expression of fimB and fimE, two genes encoding recombinases participating in controlling the orientation of the fim promoter, was decreased in BEN2908 ΔibeA. A reduction of type 1 fimbria expression due to a preferential orientation of the fim promoter in the off position was also seen in an ibeT mutant of strain BEN2908. We finally suggest a role for IbeA and IbeT in modulating the expression of type 1 fimbriae through an as yet unknown mechanism.

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